A variety of methods for the amplification of nucleic acids are known. For example, polymerase chain reaction (“PCR”) (see, e.g. U.S. Pat. No. 4,683,202) is a popular method for the amplification of nucleic acids. To successfully perform a PCR reaction, the reaction must be performed at multiple different temperatures. This requires hardware or other mechanisms for repeatedly changing the temperature of the PCR reaction. Another method for amplification of nucleic acids is referred to as loop-mediated isothermal amplification (“LAMP”) (see, e.g. U.S. Pat. No. 6,410,278). LAMP reactions may be performed isothermally, but typically involve the use of four different primers which recognize a total of six distinct sequences on the target nucleic acid.
Some amplification methods utilize DNA ligation. DNA ligation is a common molecular biology method for joining multiple DNA fragments. Ligases can seal single-strand nicks in duplex DNA, join two pieces with complementary “sticky” ends, and in some cases join two pieces with blunt, non-sticky ends. These methods find use in molecular cloning, nucleic acid diagnostics/detection, nucleic acid amplification, and related activities. Ligases and ligation methods are described, for example, in U.S. Patent Application Publication No. 20120214208; U.S. Pat. No. 7,927,853; de Lumley et al., J. Mol. Biol. (2004) 339, 1059-1075; and Rolland et al., FEMS Microbiol. Lett. (2004) 236, 267-273.
Ligation of two strands of “sticky-ended” DNA is possible, where the substrates to be joined are already pre-positioned in the case of single-strand nick sealing, and the affinity of sticky ends for each other helps drive sticky-end ligation. However, since DNA ligation depends upon the juxtaposition of the two substrates to be joined, DNA ligation is more difficult in situations where the juxtaposition of the two substrates is more difficult, or the strands are likely to become mis-aligned or to separate readily. For this reason, blunt-end and high temperature ligations are two types of ligation that are particularly difficult. Blunt-end ligation, however, depends upon random interactions of the substrates. Two adjustments in blunt-end ligation are commonly made to drive the substrate interactions: low temperature to slow down molecular motion so that random interactions last longer and thus give the ligase a better chance to join the fragments; and molecular crowding reagents such as polyethylene glycol to increase the local concentrations of substrates. Likewise, high temperature ligations are challenging because the interactions of DNA substrates are briefer. The least efficient case is thus a high temperature blunt-end ligation in which molecular crowding reagents cannot be used.
A thermostable ligase, T4 DNA ligase, has been described that can perform blunt-end ligations; however, it is inactivated at temperature above approximately 45° C., so that the range of temperature at which T4 DNA ligase is stable is relatively small. A few other examples of ligases that can be induced to join blunt-ended fragments are known, but these ligases appear to do so only in the presence of high concentrations of molecular crowding agents such as 50% polyethylene glycol (PEG). However, the utility of ligases that require such molecular crowding agents is unclear, since 50% PEG which DNA polymerization (which is required for DNA amplification).
Accordingly, in order to facilitate the generation of amplified nucleic acids for the many and growing number of applications which use amplified nucleic acids, new methods and reagents for the amplification of nucleic acids are desired.